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Abstract:

The present invention relates to the treatment of diseases and conditions
with an effective amount of a steroid having those formulas given in the
specification, or a pharmacologically-acceptable salt or ester thereof.
The disease or conditions treatable according to the invention include
angiogenic diseases and conditions of the eye, angiogenic diseases and
conditions of the brain, inflammatory diseases and conditions of the eye,
inflammatory diseases and conditions of the brain and neurodegenerative
diseases.

Claims:

1-180. (canceled)

181. A pharmaceutical product suitable for treatment of the eye
comprising a trilostane compound selected from the group consisting of
trilostane, trilostane II, trilostane III and keto-trilostane or a
pharmacologically-acceptable salt or ester thereof.

182. The product of claim 181, wherein the product is a pharmaceutical
composition formulated for treatment of the eye.

183. The composition of claim 182, wherein the composition is formulated
for intraocular injection into the eye.

184. The composition of claim 182, wherein the composition is formulated
for topical administration to the eye.

185. The composition of claim 184, wherein the composition is eye drops.

186. The composition of claim 184, wherein the composition is a topical
ophthalmic suspension.

187. The composition of claim 186, wherein the ophthalmic suspension is a
colloidal suspension.

188. The composition of claim 184, wherein the composition is a topical
ophthalmic solution.

189. The composition of claim 188, wherein the ophthalmic solution
comprises at least one of a dispersing agent, a solubilizing agent, a
wetting agent, an emulsifying agent, a pH buffering agent, a
preservative, a surfactant, a salt, a tonicity agent, and a suspending
agent.

190. The composition of claim 188, wherein the ophthalmic solution
comprises a vehicle selected from the group consisting of water, saline,
aqueous dextrose, glycerol, and ethanol.

191. The composition of claim 184, wherein the composition is a topical
ophthalmic gel.

194. The composition of claim 191, wherein the ophthalmic gel comprises a
combination of ethanol, water and propylene glycol in a ratio of
40:40:20.

195. The composition of claim 191, wherein the ophthalmic gel comprises
at least one of a gelling agent, a preservative and a tonicity agent.

196. The composition of claim 191, wherein the ophthalmic gel comprises a
gelling agent selected from the group consisting of
hydroxylethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, and ammoniated glycyrrhizinate.

197. The composition of claim 184, wherein the composition is a topical
ophthalmic hydrogel.

199. The composition of claim 198, wherein the hydrogel is a
thermoreversible hydrogel.

200. The composition of claim 184, wherein the composition is a topical
ophthalmic ointment.

201. The composition of claim 200, wherein the ophthalmic ointment
comprises a base selected from the group consisting of mineral oil,
liquid lanolin, white petrolatum, polyethylene-mineral oil gel, or a
combination thereof.

202. The product of claim 181, wherein the product is a pharmaceutical
device suitable for treatment of the eye.

203. The device of claim 202, wherein the device is a solid carrier
impregnated with the trilostane compound or pharmacologically-acceptable
salt or ester thereof.

204. The device of claim 203, wherein the device is a soft contact lens.

205. The product of claim 181, wherein the product is an ocular implant.

206. The product of any one of claims 181-205 wherein the trilostane
compound is trilostane III.

207. A method of treating an angiogenic disease or condition of the eye
comprising administering to an animal in need thereof an effective amount
of a trilostane compound selected from the group consisting of
trilostane, trilostane II, trilostane III and keto-trilostane or a
pharmacologically-acceptable salt or ester thereof.

208. The method of claim 207 wherein the disease or condition is macular
degeneration.

209. The method of claim 207 wherein the disease or condition is
neovascularization due to an ocular insult or a tumor.

210. The method of claim 207 wherein the disease or condition is
neovascular glaucoma, retinopathy of prematurity, sickle-cell retinopathy
or oxygen-induced retinopathy.

211. The method of claim 207 wherein the disease or condition is diabetic
retinopathy.

212. The method of claim 207 wherein administering comprises intraocular
injection of the trilostane compound or a pharmacologically-acceptable
salt or ester thereof into the eye.

213. The method of claim 207 wherein administering comprises topical
administration of the trilostane compound or a
pharmacologically-acceptable salt or ester thereof to the eye.

214. The method of claim 207 wherein administering comprises application
of an ointment, a gel or a hydrogel comprising the trilostane compound or
a pharmacologically-acceptable salt or ester thereof to the eye.

215. The method of claim 207 wherein administering comprises application
of a solution comprising the trilostane compound or a
pharmacologically-acceptable salt or ester thereof into the eye.

216. The method of claim 207 wherein administering comprises implantation
of an ocular implant comprising the trilostane compound or a
pharmacologically-acceptable salt or ester thereof into the eye.

217. The method of claim 207 wherein administering comprises insertion of
a pharmaceutical device into the eye suitable for treatment of the eye.

218. The method of claim 217 wherein the device is a solid carrier
impregnated with the trilostane compound or pharmacologically-acceptable
salt or ester thereof.

219. The method of claim 218 wherein the device is a soft contact lens.

220. A method of treating an angiogenic condition of the eye comprising
applying to an eye of an animal in need of such treatment, a topical
pharmaceutical composition comprising a trilostane compound selected from
the group consisting of trilostane, trilostane II, trilostane III and
keto-trilostane or pharmacologically acceptable salts and esters thereof,
wherein the composition is formulated for topical application to the eye.

221. The method of claim 220, wherein the angiogenic condition of the eye
is selected from the group consisting of macular degeneration, diabetic
retinopathy, neovascular glaucoma, retinopathy of prematurity,
sickle-cell retinopathy, oxygen-induced retinopathy, and
neovascularization due to ocular insults.

222. The method of claim 220, wherein the composition is a topical
ophthalmic suspension.

223. The method of claim 222, wherein the ophthalmic suspension is a
colloidal suspension.

224. The method of claim 220, wherein the composition is a topical
ophthalmic solution.

225. The method of claim 224, wherein the ophthalmic solution comprises
at least one of a dispersing agent, a solubilizing agent, a wetting
agent, an emulsifying agent, a pH buffering agent, a preservative, a
surfactant, a salt, a tonicity agent, and a suspending agent.

226. The method of claim 224, wherein the ophthalmic solution comprises a
vehicle selected from the group consisting of water, saline, aqueous
dextrose, glycerol, and ethanol.

227. The method of claim 220, wherein the composition is a topical
ophthalmic gel.

230. The method of claim 227, wherein the ophthalmic gel comprises a
combination of ethanol, water and propylene glycol in a ratio of
40:40:20.

231. The method of claim 227, wherein the ophthalmic gel comprises at
least one of a gelling agent, a preservative and a tonicity agent.

232. The method of claim 227, wherein the ophthalmic gel comprises a
gelling agent selected from the group consisting of
hydroxylethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, and ammoniated glycyrrhizinate.

233. The method of claim 220, wherein the composition is a topical
ophthalmic hydrogel.

[0002] The present invention relates to the treatment of diseases and
conditions with an effective amount of a steroid having those formulas
given below, or a pharmacologically-acceptable salt or ester thereof. The
disease or conditions treatable according to the invention include
angiogenic diseases and conditions of the eye or brain, inflammatory
diseases and conditions of the eye or brain, and neurodegenerative
diseases.

BACKGROUND

[0003] Over time, diabetes affects the circulatory system of the retina
causing diabetic retinopathy, which occurs in about forty percent of
diabetics. The earliest phase of this disease is known as background
diabetic retinopathy. The next stage is known as proliferative diabetic
retinopathy. In this stage, circulation problems cause areas of the
retina to become oxygen-deprived or ischemic. New, fragile vessels
develop as the circulatory system attempts to maintain adequate oxygen
levels within the retina. Unfortunately, these delicate vessels
hemorrhage easily, and blood may leak into the retina and vitreous,
causing spots or floaters, along with decreased vision. In the later
phases of the disease, continued abnormal vessel growth and scar tissue
may cause serious problems, such as retinal detachment and glaucoma.
There are several treatments for diabetic retinopathy, depending on the
stage of the disease and the specific problem that requires attention.
However, there still remains a need for additional treatments, especially
for one that is simple to administer and which has few side effects.

[0004] Age-related macular degeneration is the leading cause of severe
vision loss in people age sixty and older. There are two forms of this
disease--the dry form and the wet form. The wet form accounts for only
about fifteen percent of all cases, but it is responsible for most of the
severe vision loss that occurs in people suffering from macular
degeneration. The abnormal growth of new blood vessels (called choroidal
neovascularizations) is the cause of the severe vision loss that occurs
in wet macular degeneration. There is a dire need for an effective
treatment for wet macular degeneration, especially for one that is simple
to administer and which has few side effects.

[0005] There is also clearly a need for additional and more effective
treatments for brain tumors. Therapies that are effective for other
tumors currently have serious limitations as brain tumor treatments. The
blood brain barrier may create a particular obstacle to the effective use
of chemotherapy in the treatment of brain tumors.

[0006] Many treatments have been proposed for neurodegenerative diseases
or conditions. However, most of these treatments have not been
successful, and there remains a need for additional treatments for these
diseases.

SUMMARY OF THE INVENTION

[0007] The invention provides a method of treating an angiogenic disease
or condition of the eye. The method comprises administering to an animal
in need thereof an effective amount of a steroid of formula I or IV or a
pharmacologically-acceptable salt or ester thereof.

[0008] The invention also provides a method of treating an inflammatory
disease or condition of the eye. The method comprises administering to an
animal in need thereof an effective amount of a steroid of formula I, IV
or V or a pharmacologically-acceptable salt or ester thereof.

[0009] The invention further provides a pharmaceutical product suitable
for treatment of the eye. The product comprises a steroid of formula I,
IV or V or a pharmacologically-acceptable salt or ester thereof.
Treatment of the eye avoids systemic side effects. Particularly preferred
are topical pharmaceutical compositions because they are simple to
administer to the eye.

[0010] The invention further provides a method of treating an angiogenic
disease or condition of the brain. The method comprises administering to
an animal in need thereof an effective amount of a steroid of formula I
or IV or a pharmacologically-acceptable salt or ester thereof.

[0011] The invention also provides a method of treating an inflammatory
disease or condition of the brain. The method comprises administering to
an animal in need thereof an effective amount of a steroid of formula I,
IV or V or a pharmacologically-acceptable salt or ester thereof.

[0012] The invention further provides a method of treating a
neurodegenerative disease or condition of the brain. The method comprises
administering to an animal in need thereof an effective amount of a
steroid of formula I, IV or V or a pharmacologically-acceptable salt or
ester thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows the fluorescence measured after treatment of HUVEC
cells with trilostane III as a measure of their ability to prevent
endothelial cell invasion.

[0014] FIG. 2 shows the OD levels measured after incubation of HUVEC cells
with trilostane III as a measure of its ability to prevent initial
proliferation of endothelial cells.

[0015] FIG. 3 shows photographs of HUVEC cells taken after incubation with
trilostane III as a measure of its ability to prevent tube formation of
endothelial cells.

[0016] FIG. 4 shows the OD levels measured after incubation of HUVEC cells
with danazol as a measure of its ability to prevent initial proliferation
of endothelial cells.

[0018] FIG. 6 shows the fluorescence measured after treatment of HUVEC
cells with danazol as a measure of their ability to prevent endothelial
cell invasion.

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS OF THE
INVENTION

[0019] The invention provides a method of treating an angiogenic disease
or condition of the eye. An "angiogenic disease or condition of the eye"
is a disease or condition of the eye involving, exacerbated by, or caused
by, angiogenesis. The method comprises administering to an animal in need
thereof an amount of a steroid of formula I or IV or a
pharmacologically-acceptable salt or ester thereof that is effective to
inhibit the angiogenesis that is occurring in the eye.

[0020] Angiogenic diseases and conditions of the eye that can be treated
according to the present invention include, but are not limited to,
macular degeneration, diabetic retinopathy, neovascular glaucoma,
retinopathy of prematurity, sickle-cell retinopathy, oxygen-induced
retinopathy, neovascularization due to ocular insults (such as traumatic
or surgical injury or transplantation of eye tissue) and tumors.

[0021] The invention also provides a method of treating an angiogenic
disease or condition of the brain. An "angiogenic disease or condition of
the brain" is a disease or condition of the brain involving, exacerbated
by, or caused by, angiogenesis. The method comprises administering to an
animal in need thereof an amount of a steroid of formula I or IV or a
pharmacologically-acceptable salt or ester thereof that is effective to
inhibit the angiogenesis that is occurring in the brain.

[0022] Angiogenic diseases and conditions of the brain that can be treated
according to the present invention include, but are not limited to,
tumors, neovascularization due to brain damage (such as caused by
traumatic or surgical injury of brain tissue or stroke).

[0023] Brain tumors that can be treated according to the present invention
include, but are not limited to, any benign or cancerous tumor, including
primary tumors and metastatic (secondary) tumors.

[0024] About half of all primary brain tumors are gliomas. Gliomas include
astrocytomas (e.g., pilocytic astrocytomas, low-grade astrocytomas,
anaplastic (high-grade) astrocytomas and glioblastomas multiforme), brain
stem gliomas ependymomas, ganglioneuromas, juvenile pilocytic gliomas,
mixed gliomas, oligodendrogliomas and optic nerve gliomas. Glioblastomas
are the most common malignant brain tumors in adults and are probably the
most resistant of all cancers to treatment.

[0025] Meningiomas account for about 27% of primary brain tumors, and most
are benign. However, unlike benign tumors elsewhere, benign brain tumors
can sometimes cause disability and may sometimes be life threatening.
Meningiomas are often curable with surgery, but treatment according to
the present invention can be used instead of or in addition to surgery.

[0027] Metastatic brain tumors are tumors that have spread to the brain
from another part of the body. The most common cancers that metastasize
to the brain include breast, melanoma and lung cancers. Metastatic brain
tumors are the most common form of brain tumor and considerably outnumber
primary brain tumors.

[0028] The invention further provides a method of treating an inflammatory
disease or condition of the eye. An "inflammatory disease or condition of
the eye" is a disease or condition of the eye involving, exacerbated by,
or caused by, inflammation. The method comprises administering to an
animal in need thereof an amount of a steroid of formula I, IV or V or a
pharmacologically-acceptable salt or ester thereof that is effective to
inhibit the inflammation that is occurring in the eye.

[0029] Inflammatory diseases and conditions of the eye that can be treated
according to the present invention include, but are not limited to,
uveitis, scleritis, keratitis, retinitis, iritis, uveoretinitis,
uveoscleritis, conjunctivitis, Mooren's ulcer and inflammatory ocular
manifestations in allergies and in arthritic, rheumatic and connective
tissue diseases (see, e.g., Bucknall, Rheumatology, 44(10):1207-1209
(2005).

[0030] The invention also provides a method of treating an inflammatory
disease or condition of the brain. An "inflammatory disease or condition
of the brain" is a disease or condition of the brain involving,
exacerbated by, or caused by, inflammation. The method comprises
administering to an animal in need thereof an amount of a steroid of
formula I, IV or V or a pharmacologically-acceptable salt or ester
thereof that is effective to inhibit the inflammation that is occurring
in the brain.

[0031] Inflammatory diseases and conditions of the brain that can be
treated according to the present invention include, but are not limited
to, abscesses (including abscesses caused by bacterial, fungal and
parasitic infections), meningitis (including bacterial meningitis,
tuberculosis and sarcoidosis), encephalitis (including Herpes simplex
encephalitis, Eastern and Western equine encephalitis, St. Louis
encephalitis, California virus encephalitis, Lyme disease and
post-infectious encephalitis), vasculitis, autism and neurodegenerative
diseases.

[0032] Neurodegenerative diseases that can be treated according to the
present invention include, but are not limited to, Alzheimer's disease,
amyotrophic lateral sclerosis, Huntington's chorea, multiple sclerosis,
Parkinson's disease and senile dementia.

[0033] "Treat," "treating" or "treatment" is used herein to mean to reduce
(wholly or partially) the symptoms, duration or severity of a disease or
condition, including curing the disease, or to prevent the disease or
condition.

[0034] "Inhibit" or "inhibiting" is used herein to mean to reduce (wholly
or partially) or to prevent.

[0035] "Angiogenesis" means the development of new blood vessels.
"Angiogenesis" is also used herein to mean the same as, or to include,
neovascularization, vascularization, arterialization and vasculogenesis.

[0036] As noted above, the steroids that can be used in the practice of
the present invention are those compounds of formulas I, IV and V shown
below and pharmacologically acceptable salts and esters thereof.

[0037] Formula I is:

##STR00001##

In Formula I:

[0038] R1 is an alkyl group having from 1 to 6 carbon atoms, an
alkenyl group having from 2 to 6 carbon atoms or an alkynyl group having
from 2 to 6 carbon atoms;

[0039] R2 is hydroxyl, an alkoxy group having from 1 to 6 carbon
atoms, an alkanoyloxy group having from 1 to 7 carbon atoms, a group of
formula (II) or a group of formula (III):

##STR00002##

[0040] wherein R11 is hydrogen, an alkyl group having from 1 to 6
carbon atoms, a hydroxyl group, an alkoxy group having from 1 to 6 carbon
atoms or a group of formula --N(R14)2 wherein each group
R14 is the same or different and is hydrogen or an alkyl group
having from 1 to 6 carbon atoms,

[0041] each of R12 and R13 is the same or different and is
hydrogen or an alkyl group having from 1 to 6 carbon atoms, and

[0042] m and n are the same or different and each is 0 or an integer of
from 1 to 4;

[0043] each of R3 and R4 is the same or different and is
hydrogen or an alkyl group having from 1 to 6 carbon atoms;

[0044] each of R5 and R6 is the same or different and is
hydrogen or an alkyl group having from 1 to 6 carbon atoms or R5 and
R6 together represent a single bond; and

[0045] R7, R8, R9 and R10 together with the carbon
atoms to which they are attached represent a 5- to 9-membered
heterocyclic group, said 5- to 9-membered heterocyclic group optionally
being substituted with from 1 to 7 substituents (said substituents are
the same or different and are selected from substituent group α
defined below).

[0046] Substituent group α is a group consisting of a halogen atom,
a hydroxyl group, a cyano group, an amino group, an alkyl group having
from 1 to 6 carbon atoms, an alkoxy group having from 1 to 6 carbon
atoms, an alkylthio group having from 1 to 6 carbon atoms, an
alkylsulfinyl group having from 1 to 6 carbon atoms, an alkylsulfonyl
group having from 1 to 6 carbon atoms, a phenyl group and a group of
formula --N(R16)2 wherein each group R16 is the same or
different and is hydrogen or an alkyl group having from 1 to 6 carbon
atoms.

[0047] Where R7, R8, R9 and R16 together with the
carbon atoms to which they are attached represent an optionally
substituted 5- to 9-membered heterocyclic group, said heterocyclic group
is a 5- to 9-membered heterocyclic group containing from 1 to 4 atoms
selected from a group consisting of a nitrogen atom, an oxygen atom and a
sulfur atom and may be, for example, an unsaturated heterocyclic group
such as a furyl group, a thienyl group, a pyrrolyl group, an azepinyl
group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an
isoxazolyl group, a thiazolyl group, an isothiazolyl group, a
1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a
thiadiazolyl group, a pyranyl group, a pyridyl group, a pyridazinyl
group, a pyrimidinyl group, a pyrazinyl group, an azepinyl group, an
azocinyl group or an azoninyl group; or a group wherein the unsaturated
heterocyclic groups described above are partially or completely reduced,
such as a morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl
group, a pyrrolinyl group, a imidazolidinyl group, a imidazolinyl group,
a pyrazolidinyl group, a pyrazolinyl group, a piperidyl group, a
piperazinyl group, a perhydroazepinyl group, a perhydroazocinyl group or
a perhydroazoninyl group; preferably it is a 5- to 7-membered
heterocyclic group containing one or more nitrogen atom and optionally
containing an oxygen atom and/or a sulfur atom, which is, for example, an
unsaturated heterocyclic group such as a pyrrolyl group, an azepinyl
group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an
isoxazolyl group, a thiazolyl group, an isothiazolyl group, a
1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a
thiadiazolyl group, a pyridyl group, a pyridazinyl group, a pyrimidinyl
group or a pyrazinyl group; or a group wherein this unsaturated
heterocyclic group is partially or completely reduced, such as a
morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl group, a
pyrrolinyl group, a imidazolidinyl group, a imidazolinyl group, a
pyrazolidinyl group, a pyrazolinyl group, a piperidyl group or a
piperazinyl group; and more preferably it is an isoxazolyl group or a
pyrazolyl group for R7, R8, R9 and R10 together with
the carbon atoms to which they are attached.

[0048] Formula IV is:

##STR00003##

In Formula IV:

[0049] R18, R19 and R21 are the same or different and each
is hydrogen or an alkyl group having from 1 to 6 carbon atoms;

[0050] R17 is hydrogen, an alkyl group having from 1 to 6 carbon
atoms or an alkenyl group having from 2 to 6 carbon atoms,

[0051] R22 is hydroxyl, an alkoxy group having from 1 to 6 carbon
atoms, an alkanoyloxy group having from 1 to 7 carbon atoms, a group of
formula (II) as defined above for formula (I) or a group of formula (III)
as defined above for formula (I), or

[0052] R17 and R22 together represent an oxo group, an
ethylenedioxy group or a propylenedioxy group;

[0053] each of R20 and R24 is the same or different and is
hydrogen or an alkyl group having from 1 to 6 carbon atoms or R20
and R24 together represent a single bond;

[0054] each of R23 and R29 is the same or different and is
hydrogen or an alkyl group having from 1 to 6 carbon atoms or R23
and R29 together represent an epoxy linkage;

[0055] each of R25, R26, R27 and R28 is the same or
different and is hydrogen, an alkyl group having from 1 to 6 carbon
atoms, a cyano group, a hydroxyl group, an alkoxy group having from 1 to
6 carbon atoms or a group of formula --N(R30)2 wherein each
group R30 is the same or different and is hydrogen or an alkyl group
having from 1 to 6 carbon atoms, or

[0056] R25 and R26 and/or R27 and R28 together with
the carbon atom to which they are attached represent a carbonyl group, or

[0057] R25, R26, R27 and R28 together with the carbon
atoms to which they are attached represent a 5- to 9-membered
heterocyclic group, said 5- to 9-membered heterocyclic group optionally
being substituted with from 1 to 7 substituents (said substituents are
the same or different and are selected from substituent group α
defined above).

[0058] Where R25, R26, R27 and R28 together with the
carbon atoms to which they are attached represent an optionally
substituted 5- to 9-membered heterocyclic group, said heterocyclic group
is a 5- to 9-membered heterocyclic group containing from 1 to 4 atoms
selected from a group consisting of a nitrogen atom, an oxygen atom and a
sulfur atom and may be, for example, an unsaturated heterocyclic group
such as a furyl group, a thienyl group, a pyrrolyl group, an azepinyl
group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an
isoxazolyl group, a thiazolyl group, an isothiazolyl group, a
1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a
thiadiazolyl group, a pyranyl group, a pyridyl group, a pyridazinyl
group, a pyrimidinyl group, a pyrazinyl group, an azepinyl group, an
azocinyl group or an azoninyl group; or a group wherein the unsaturated
heterocyclic groups described above are partially or completely reduced,
such as a morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl
group, a pyrrolinyl group, a imidazolidinyl group, a imidazolinyl group,
a pyrazolidinyl group, a pyrazolinyl group, a piperidyl group, a
piperazinyl group, a perhydroazepinyl group, a perhydroazocinyl group or
a perhydroazoninyl group; preferably it is a 5- to 7-membered
heterocyclic group containing one or more nitrogen atom and optionally
containing an oxygen atom and/or a sulfur atom, which is, for example, an
unsaturated heterocyclic group such as a pyrrolyl group, an azepinyl
group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an
isoxazolyl group, a thiazolyl group, an isothiazolyl group, a
1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a
thiadiazolyl group, a pyridyl group, a pyridazinyl group, a pyrimidinyl
group or a pyrazinyl group; or a group wherein this unsaturated
heterocyclic group is partially or completely reduced, such as a
morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl group, a
pyrrolinyl group, a imidazolidinyl group, a imidazolinyl group, a
pyrazolidinyl group, a pyrazolinyl group, a piperidyl group or a
piperazinyl group; and more preferably it is an isoxazolyl group for
R25, R26, R27 and R28 together with the carbon atoms
to which they are attached.

[0059] Formula V is:

##STR00004##

In Formula V:

[0060] R35 is an alkyl group having from 1 to 6 carbon atoms, an
alkenyl group having from 2 to 6 carbon atoms or an alkynyl group having
from 2 to 6 carbon atoms;

[0061] R36 is hydroxyl, an alkoxy group having from 1 to 6 carbon
atoms, an alkanoyloxy group having from 1 to 7 carbon atoms, a group of
formula (II) as defined above for formula (I) or a group of formula (III)
as defined above for formula (I), or

[0062] each of R37 and R38 is the same or different and is
hydrogen or an alkyl group having from 1 to 6 carbon atoms;

[0063] each of R39 and R40 is the same or different and is
hydrogen or an alkyl group having from 1 to 6 carbon atoms or R39
and R40 together represent a single bond;

[0064] each of R41, R42, R43 and R44 is the same or
different and is hydrogen, an alkyl group having from 1 to 6 carbon
atoms, a hydroxyl group, an alkoxy group having from 1 to 6 carbon atoms
or a group of formula --N(R45)2 wherein each group R45 is
the same or different and is hydrogen or an alkyl group having from 1 to
6 carbon atoms, or

[0065] R41 and R42 and/or R43 and R44 together with
the carbon atom to which they are attached represent a carbonyl group, or

[0066] R41, R42, R43 and R44 together with the carbon
atoms to which they are attached represent a 5- to 9-membered
heterocyclic group, said 5- to 9-membered heterocyclic group optionally
being substituted with from 1 to 7 substituents, which may be the same or
different and are selected from substituent group α defined above.

[0067] Where R41, R42, R43 and R44 together with the
carbon atoms to which they are attached represent an optionally
substituted 5- to 9-membered heterocyclic group, said heterocyclic group
is a 5- to 9-membered heterocyclic group containing from 1 to 4 atoms
selected from a group consisting of a nitrogen atom, an oxygen atom and a
sulfur atom and may be, for example, an unsaturated heterocyclic group
such as a furyl group, a thienyl group, a pyrrolyl group, an azepinyl
group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an
isoxazolyl group, a thiazolyl group, an isothiazolyl group, a
1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a
thiadiazolyl group, a pyranyl group, a pyridyl group, a pyridazinyl
group, a pyrimidinyl group, a pyrazinyl group, an azepinyl group, an
azocinyl group or an azoninyl group; or a group wherein the unsaturated
heterocyclic groups described above are partially or completely reduced,
such as a morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl
group, a pyrrolinyl group, a imidazolidinyl group, a imidazolinyl group,
a pyrazolidinyl group, a pyrazolinyl group, a piperidyl group, a
piperazinyl group, a perhydroazepinyl group, a perhydroazocinyl group or
a perhydroazoninyl group; preferably it is a 5- to 7-membered
heterocyclic group containing one or more nitrogen atom and optionally
containing an oxygen atom and/or a sulfur atom, which is, for example, an
unsaturated heterocyclic group such as a pyrrolyl group, an azepinyl
group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an
isoxazolyl group, a thiazolyl group, an isothiazolyl group, a
1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a
thiadiazolyl group, a pyridyl group, a pyridazinyl group, a pyrimidinyl
group or a pyrazinyl group; or a group wherein this unsaturated
heterocyclic group is partially or completely reduced, such as a
morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl group, a
pyrrolinyl group, a imidazolidinyl group, a imidazolinyl group, a
pyrazolidinyl group, a pyrazolinyl group, a piperidyl group or a
piperazinyl group; and more preferably it is an isoxazolyl group or a
R44 pyrazolyl group for R41, R42, R43 and R44
together with the carbon atoms to which they are attached.

[0068] The alkyl group having from 1 to 6 carbon atoms is a straight or
branched chain alkyl group having from 1 to 6 carbon atoms and may be,
for example, a methyl group, an ethyl group, a propyl group, an isopropyl
group, a butyl group, an isobutyl group, an s-butyl group, a t-butyl
group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl
group, a 1-methylbutyl group, a hexyl group, a 1-methylpentyl group, a
2-methylpentyl group, a 3-methylpentyl group, a 1-ethylbutyl group or a
2-ethylbutyl group; preferably it is an alkyl group having from 1 to 4
carbon atoms such as a methyl group, an ethyl group, a propyl group, an
isopropyl group, a butyl group, an isobutyl group, an s-butyl group or a
t-butyl group; more preferably it is a methyl group, an ethyl group, a
propyl group or an isopropyl group; and most preferably it is a methyl
group.

[0069] The alkenyl group having from 2 to 6 carbon atoms is a straight or
branched chain alkenyl group having from 2 to 6 carbon atoms and may be,
for example, vinyl, 2-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl,
2-ethyl-2-propenyl, 2-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl,
1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl,
1-ethyl-3-butenyl, 2-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl,
3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl,
1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl
and 5-hexenyl groups. Alkenyl groups having from 2 to 4 carbon atoms are
preferred, and alkenyl groups having 2 or 3 carbon atoms are most
preferred.

[0070] The alkynyl group having from 2 to 6 carbon atoms is a straight or
branched chain alkynyl group having from 2 to 6 carbon atoms and may be,
for example, ethynyl, 2-propynyl, 1-methyl-2-propynyl, 2-butynyl,
1-methyl-2-butynyl, 1-ethyl-2-butynyl, 3-butynyl, 1-methyl-3-butynyl,
2-methyl-3-butynyl, 1-ethyl-3-butynyl, 2-pentynyl, 1-methyl-2-pentynyl,
3-pentynyl, 1-methyl-3-pentynyl, 2-methyl-3-pentynyl, 4-pentynyl,
1-methyl-4-pentynyl, 2-methyl-4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl
and 5-hexynyl groups. Alkynyl groups having from 2 to 4 carbon atoms are
preferred, and alkynyl groups having 2 or 3 carbon atoms are most
preferred.

[0071] The alkanoyloxy group having from 1 to 7 carbon atoms is a
carbonyloxy group (--COO--) the carbon atom of which is substituted with
a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms as
described above and may be, for example, a formyloxy group, an acetyloxy
group, a propionyloxy group, a butyryloxy group, an isobutyryloxy group,
a pentanoyloxy group or a hexanoyloxy group; it is preferably an
alkanoyloxy group having from 2 to 5 carbon atoms such as an acetyloxy
group, a propionyloxy group, a butyryloxy group or an isobutyryloxy
group; and more preferably it is an acetyloxy group.

[0072] The alkoxy group having from 1 to 6 carbon atoms is a hydroxy group
in which the hydrogen atom is substituted with an alkyl group having from
1 to 6 carbon atoms as described above and may be, for example, a methoxy
group, an ethoxy group, a n-propoxy group, an isopropoxy group, a
n-butoxy group, an isobutoxy group, an s-butoxy group, a tert-butoxy
group, an n-pentyloxy group, an isopentyloxy group, a 2-methylbutoxy
group, a neopentyloxy group, an n-hexyloxy group, a 4-methylpentyloxy
group, a 3-methylpentyloxy group, a 2-methylpentyloxy group, a
3,3-dimethylbutoxy group, a 2,2-dimethylbutoxy group, a
1,1-dimethylbutoxy group, a 1,2-dimethylbutoxy group, a
1,3-dimethylbutoxy group or a 2,3-dimethylbutoxy group; it is preferably
an alkoxy group having from 1 to 4 carbon atoms such as a methoxy group,
an ethoxy group, an n-propoxy group or an n-butoxy group; and more
preferably it is a methoxy group.

[0073] The alkylthio group having from 1 to 6 carbon atoms is a mercapto
group substituted with an alkyl group having from 1 to 6 carbon atoms as
described above and may be, for example, a methylthio group, an ethylthio
group, an n-propylthio group, an isopropylthio group, an n-butylthio
group, an isobutylthio group, an s-butylthio group, a tert-butylthio
group, an n-pentylthio group, an isopentylthio group, a 2-methylbutylthio
group, a neopentylthio group, a 1-ethylpropylthio group, an n-hexylthio
group, an isohexylthio group, a 4-methylpentylthio group, a
3-methylpentylthio group, a 2-methylpentylthio group, a
1-methylpentylthio group, a 3,3-dimethylbutylthio group, a
2,2-dimethylbutylthio group, a 1,1-dimethylbutylthio group, a
1,2-dimethylbutylthio group, a 1,3-dimethylbutylthio group, a
2,3-dimethylbutylthio group or a 2-ethylbutylthio group; it is preferably
an alkylthio group having from 1 to 4 carbon atoms such as a methylthio
group, an ethylthio group, an n-propylthio group or an n-butylthio group;
and more preferably it is a methylthio group.

[0074] The alkylsulfinyl group having from 1 to 6 carbon atoms is a
sulfinyl group (--SO--) which is substituted with an alkyl group having
from 1 to 6 carbon atoms as described above and may be, for example, a
methanesulfinyl group, an ethanesulfinyl group, an n-propanesulfinyl
group, an isopropanesulfinyl group, an n-butanesulfinyl group, an
isobutanesulfinyl group, an s-butanesulfinyl group, a tert-butanesulfinyl
group, an n-pentanesulfinyl group, an isopentanesulfinyl group, a
2-methylbutanesulfinyl group, a neopentanesulfinyl group, an
n-hexanesulfinyl group, a 4-methylpentanesulfinyl group, a
3-methylpentanesulfinyl group, a 2-methylpentanesulfinyl group, a
3,3-dimethylbutanesulfinyl group, a 2,2-dimethylbutanesulfinyl group, a
1,1-dimethylbutanesulfinyl group, a 1,2-dimethylbutanesulfinyl group, a
1,3-dimethylbutanesulfinyl group or a 2,3-dimethylbutanesulfinyl group;
preferably it is an alkylsulfinyl group having from 1 to 4 carbon atoms
such as a methanesulfinyl group, an ethanesulfinyl group, an
n-propanesulfinyl group, an isopropanesulfinyl group or an
n-butanesulfinyl group; and more preferably it is a methanesulfinyl
group.

[0075] The alkylsulfonyl group having from 1 to 6 carbon atoms is a
sulfonyl group (--SO2--) substituted with an alkyl group having from
1 to 6 carbon atoms as described above and may be, for example, a
methanesulfonyl group, an ethanesulfonyl group, an n-propanesulfonyl
group, an isopropanesulfonyl group, an n-butanesulfonyl group, an
isobutanesulfonyl group, an s-butanesulfonyl group, a tert-butanesulfonyl
group, an n-pentanesulfonyl group, an isopentanesulfonyl group, a
2-methylbutanesulfonyl group, a neopentanesulfonyl group, an
n-hexanesulfonyl group, a 4-methylpentanesulfonyl group, a
3-methylpentanesulfonyl group, a 2-methylpentanesulfonyl group, a
3,3-dimethylbutanesulfonyl group, a 2,2-dimethylbutanesulfonyl group, a
1,1-dimethylbutanesulfonyl group, a 1,2-dimethylbutanesulfonyl group, a
1,3-dimethylbutanesulfonyl group or a 2,3-dimethylbutanesulfonyl group;
preferably it is an alkylsulfonyl group having from 1 to 4 carbon atoms
such as a methanesulfonyl group, an ethanesulfonyl group, an
n-propanesulfonyl group or an n-butanesulfonyl group; and more preferably
it is a methanesulfonyl group.

[0076] Methods of making the steroids of formulas (I), (IV) and (V) are
known in the art. See e.g., U.S. Pat. Nos. 3,135,743, 3,296,255 and GB
1,123,770 and 2,130,588. Also, danazol, trilostane and other compounds
covered by formulas (I), (IV) and (V) are available commercially from,
e.g., LKT Laboratories Inc., Mochida Pharmaceuticals, Sanofi Inc. and
Sanofi Winthrop.

[0077] Where the compound of formula (I), (IV) or (V) of the present
invention or a pharmacologically acceptable ester thereof has a basic
group, the compound can be converted to a salt by reacting it with an
acid, and in the case where the compound of formula (I), (IV) or (V) of
the present invention or a pharmacologically acceptable ester thereof has
an acidic group, the compound can be converted to a salt by reacting it
with a base. The compounds of the present invention encompass such salts.
Where said salts are to be used for a therapeutic use, they must be
pharmacologically acceptable.

[0078] Preferred examples of the salts formed with a basic group present
in the compound of formula (I), (IV) or (V) or a pharmacologically
acceptable ester thereof include inorganic acid salts such as
hydrohalogenated acid salts (e.g. hydrochlorides, hydrobromides and
hydroiodides), nitrates, perchlorates, sulfates and phosphates; organic
acid salts such as lower alkanesulfonates in which the lower alkyl moiety
thereof is an alkyl group having from 1 to 6 carbon atoms as defined
above (e.g. methanesulfonates, trifluoromethanesulfonates and
ethanesulfonates), arylsulfonates in which the aryl moiety thereof is an
aryl group having from 6 to 14 carbon atoms (e.g. benzenesulfonate or
p-toluenesulfonate), acetates, malates, fumarates, succinates, citrates,
ascorbates, tartrates, oxalates and maleates; and amino acid salts such
as glycine salts, lysine salts, arginine salts, ornithine salts,
glutamates and aspartates. Hydrohalogenated acid salts are particularly
preferred.

[0080] The compounds of formulas (I), (IV) and (V) of the present
invention and pharmacologically acceptable salts and esters thereof of
the present invention can sometimes take up water upon exposure to the
atmosphere or when recrystallized to absorb water or to form a hydrate
and such hydrates are also included within the scope of the present
invention. Additionally, certain other solvents may be taken up by the
compounds of the present invention to produce solvates, which also form a
part of the present invention.

[0081] The compounds of formulas (I), (IV) and (V) of the present
invention sometimes contain one or more asymmetric centres, and can
therefore faun optical isomers (including diastereoisomers). For the
compounds of the present invention, each of said isomers and mixture of
said isomers are depicted by a single formula, i.e., the formula (I),
(IV) or (V) respectively. Accordingly, the present invention covers both
the individual isomers and mixtures thereof in any proportion, including
racemic mixtures.

[0082] The present invention encompasses esters of the compounds of
formulas (I), (IV) and (V). These esters are compounds of formulas (I),
(IV) and (V) in which a hydroxyl group or a carboxy group of said
compound of formula (I), (IV) or (V) is modified by the addition of a
protecting group using conventional techniques well-known in the art
(see, for example, "Protective Groups in Organic Synthesis, Second
Edition, Theodora W. Greene and Peter G. M. Wuts, 1991, John Wiley &
Sons, Inc.).

[0083] There is no particular restriction on the nature of this protecting
group, provided that, where the ester is intended for therapeutic
purposes, it must be pharmacologically acceptable, i.e. the protecting
group must be capable of being removed by a metabolic process (e.g.
hydrolysis) on administration of said compound to the body of a live
mammal to give a compound of formula (I), (IV) or (V) or a salt thereof.
In other words, the pharmacologically acceptable esters are pro-drugs of
the compounds of formula (I), (IV) or (V) of the present invention.

[0084] Whether an ester of a compound of formula (I), (IV) or (V) of the
present invention is pharmacologically acceptable can be easily
determined. The compound under investigation is intravenously
administered to an experimental animal such as a rat or mouse and the
body fluids of the animal are thereafter studied. If a compound of
formula (I), (IV) or (V) or a pharmacologically acceptable salt thereof
can be detected in the body fluids, the compound under investigation is
judged to be a pharmacologically acceptable ester.

[0085] The compounds of formula (I), (IV) or (V) of the present invention
can be converted to an ester, examples of which include a compound of
formula (I), (IV) or (V) in which a hydroxyl group present therein is
esterified. The ester residue must be capable of being removed by a
metabolic process (e.g. hydrolysis) in vivo in order for the esterified
compound to be one which is pharmacologically acceptable. Preferred
examples of such a protecting group include the following: [0086] (i)
1-(acyloxy)lower alkyl groups, examples of which include: [0087]
1-(aliphatic acyloxy)lower alkyl groups which comprise an alkyl group
having from 1 to 6 carbon atoms as defined above which is substituted
with an alkylcarbonyloxy group having from 1 to 6 carbon atoms, examples
of which include formyloxymethyl, acetoxymethyl, propionyloxymethyl,
butyryloxymethyl, pivaloyloxymethyl, valeryloxymethyl,
isovaleryloxymethyl, hexanoyloxymethyl, 1-formyloxyethyl, 1-acetoxyethyl,
1-propionyloxyethyl, 1-butyryloxyethyl, 1-pivaloyloxyethyl,
1-valeryloxyethyl, 1-isovaleryloxyethyl, 1-hexanoyloxyethyl,
1-formyloxypropyl, 1-acetoxypropyl, 1-propionyloxypropyl,
1-butyryloxypropyl, 1-pivaloyloxypropyl, 1-valeryloxypropyl,
1-isovaleryloxypropyl, 1-hexanoyloxy-propyl, 1-acetoxybutyl,
1-propionyloxybutyl, 1-butyryloxybutyl, 1-pivaloyloxybutyl,
1-acetoxypentyl, 1-propionyloxypentyl, 1-butyryloxypentyl,
1-pivaloyloxypentyl and 1-pivaloyloxyhexyl groups, [0088]
1-(cycloalkylcarbonyloxy)lower alkyl groups which comprise an alkyl group
having from 1 to 6 carbon atoms as defined above which is substituted
with a cycloalkylcarbonyloxy group in which a carbonyloxy group is
substituted with a cycloalkyl group having from 1 to 6 carbon atoms,
examples of which include cyclopentylcarbonyloxymethyl,
cyclohexylcarbonyloxy-methyl, 1-cyclopentylcarbonyloxyethyl,
1-cyclohexylcarbonyloxyethyl, 1-cyclopentylcarbonyloxypropyl,
1-cyclohexylcarbonyloxypropyl, 1-cyclopentyl-carbonyloxybutyl and
1-cyclohexylcarbonyloxybutyl groups, and [0089] 1-(aromatic acyloxy)lower
alkyl groups which comprise an alkyl group having from 1 to 6 carbon
atoms as defined above which is substituted with an arylcarbonyloxy group
which comprises an oxygen atom which is substituted with an arylcarbonyl
group, examples of which include benzoyloxymethyl groups; [0090] (ii)
substituted carbonyloxyalkyl groups, examples of which include: [0091]
(lower alkoxycarbonyloxy)alkyl groups which comprise an alkyl group
having from 1 to 6 carbon atoms as defined above or a cycloalkyl group
having from 1 to 6 carbon atoms which is substituted with a lower
alkoxycatbonyloxy group which comprises a carbonyloxy group substituted
with an alkoxy group having from 1 to 6 carbon atoms as defined above or
a cycloalkoxy group having from 1 to 6 carbon atoms, examples of which
include methoxycarbonyloxymethyl, ethoxycarbonyloxymethyl,
propoxy-carbonyloxymethyl, isopropoxycarbonyloxymethyl,
butoxycarbonyloxymethyl, isobutoxycarbonyloxymethyl,
pentyloxycarbonyloxymethyl, hexyloxycarbonyloxy-methyl,
cyclohexyloxycarbonyloxymethyl,
cyclohexyloxycarbonyloxy(cyclohexyl)-methyl, 1-(methoxycarbonyloxy)ethyl,
1-(ethoxycarbonyloxy)ethyl, 1-(propoxy-carbonyloxy)ethyl,
1-(isopropoxycarbonyloxy)ethyl, 1-(butoxycarbonyloxy)ethyl,
1-(isobutoxycarbonyloxy)ethyl, 1-(t-butoxycarbonyloxy)ethyl,
1-(pentyloxy-carbonyloxy)ethyl, 1-(hexyloxycarbonyloxy)ethyl,
1-(cyclopentyloxycarbonyloxy)-ethyl, 1-(cyclopentyloxycarbonyloxy)propyl,
1-(cyclohexyloxycarbonyloxy)propyl, 1-(cyclopentyloxycarbonyloxy)butyl,
1-(cyclohexyloxycarbonyloxy)butyl, 1-(cyclohexyloxycarbonyloxy)ethyl,
1-(ethoxycarbonyloxy)propyl, 1-(methoxy-carbonyloxy)propyl,
1-(ethoxycarbonyloxy)propyl, 1-(propoxycarbonyloxy)propyl,
1-(isopropoxycarbonyloxy)propyl, 1-(butoxycarbonyloxy)propyl,
1-(isobutoxy-carbonyloxy)propyl, 1-(pentyloxycarbonyloxy)propyl,
1-(hexyloxycarbonyloxy)-propyl, 1-(methoxycarbonyloxy)butyl,
1-(ethoxycarbonyloxy)butyl, 1-(propoxy-carbonyloxy)butyl,
1-(isopropoxycarbonyloxy)butyl, 1-(butoxycarbonyloxy)butyl,
1-(isobutoxycarbonyloxy)butyl, 1-(methoxycarbonyloxy)pentyl,
1-(ethoxy-carbonyloxy)pentyl, 1-(methoxycarbonyloxy)hexyl and
1-(ethoxycarbonyloxy)hexyl groups, and oxodioxolenylmethyl groups, which
comprise a methyl group which is substituted with an oxodioxolenyl group
which itself may optionally be substituted with a group selected from the
group consisting of alkyl groups having from 1 to 6 carbon atoms as
defined above and aryl groups having from 6 to 14 carbon atoms as defined
above which may optionally be substituted with at least one alkyl group
having from 1 to 6 carbon atoms as defined above, alkoxy group having
from 1 to 6 carbon atoms as defined above or a halogen atom, examples of
which include (5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl,
[5-(4-methylphenyl)-2-oxo-1,3-dioxolen-4-yl]methyl,
[5-(4-methoxyphenyl)-2-oxo-1,3-dioxolen-4-yl]methyl,
[5-(4-fluorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl,
[5-(4-chlorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl,
(2-oxo-1,3-dioxolen-4-yl)-methyl,
(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl,
(5-ethyl-2-oxo-1,3-dioxolen-4-yl)methyl,
(5-propyl-2-oxo-1,3-dioxolen-4-yl)methyl,
(5-isopropyl-2-oxo-1,3-dioxolen-4-yl)methyl and
(5-butyl-2-oxo-1,3-dioxolen-4-yl)methyl groups; [0092] (iii) phthalidyl
groups which comprise a phthalidyl group which may optionally be
substituted with a substituent selected from the group consisting of
alkyl groups having from 1 to 6 carbon atoms as defined above and alkoxy
groups having from 1 to 6 carbon atoms as defined above, examples of
which include phthalidyl, dimethylphthalidyl and dimethoxyphthalidyl
groups; [0093] (iv) aliphatic acyl groups, examples of which include:
[0094] alkylcarbonyl groups having from 1 to 25 carbon atoms, examples of
which include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl,
pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl,
3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl,
3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl,
pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl,
14-methyl-pentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl,
15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl,
eicosanoyl and heneicosanoyl groups, [0095] ester forming residues of a
saturated or unsaturated C2-C10 aliphatic di-carboxylic acids
such as a fumarate, a maleate, oxalate, malonate or succinate, [0096]
halogenated alkylcarbonyl groups having from 1 to 25 carbons in which the
alkyl moiety thereof is substituted by at least one halogen atom,
examples of which include chloroacetyl, dichloroacetyl, trichloroacetyl
and trifluoroacetyl groups, [0097] lower alkoxyalkylcarbonyl groups which
comprise an alkylcarbonyl group having from 1 to 25 carbon atoms in which
the alkyl moiety thereof is substituted with at least one C1-C6
alkoxy group as defined above, examples of said lower alkoxyalkylcarbonyl
groups including methoxyacetyl groups, and [0098] unsaturated
alkylcarbonyl groups having from 1 to 25 carbon atoms, examples of which
include acryloyl, propioloyl, methacryloyl, crotonoyl, isocrotonoyl and
(E)-2-methyl-2-butenoyl groups; [0099] of these, alkylcarbonyl groups
having from 1 to 6 carbon atoms are preferred; [0100] (v) aromatic acyl
groups, examples of which include: [0101] arylcarbonyl groups which
comprise a carbonyl group which is substituted with an aryl group having
from 6 to 14 carbon atoms as defined above, examples of which include
benzoyl, α-naphthoyl and β-naphthoyl groups, [0102]
halogenated arylcarbonyl groups which comprise an arylcarbonyl group as
defined above which is substituted with at least one halogen atom,
examples of which include 2-bromobenzoyl, 4-chlorobenzoyl and
2,4,6-trifluorobenzoyl groups, [0103] lower alkylated arylcarbonyl groups
which comprise an arylcarbonyl group as defined above which is
substituted with at least one alkyl group having from 1 to 6 carbon atoms
as defined above, examples of which include 2,4,6-trimethyl-benzoyl and
4-toluoyl groups, [0104] lower alkoxylated arylcarbonyl groups which
comprise an arylcarbonyl group as defined above which is substituted with
at least one alkoxy group having from 1 to 6 carbon atoms as defined
above, examples of which include 4-anisoyl groups, [0105] nitrated
arylcarbonyl groups which comprise an arylcarbonyl group as defined above
which is substituted with at least one nitro group, examples of which
include 4-nitrobenzoyl and 2-nitrobenzoyl groups, [0106] lower
alkoxycarbonylated arylcarbonyl groups which comprise an arylcarbonyl
group as defined above which is substituted with a carbonyl group which
is itself substituted with an alkoxy group having from 1 to 6 carbon
atoms as defined above, examples of which include
2-(methoxycarbonyl)benzoyl groups, and [0107] arylated arylcarbonyl
groups which comprise an arylcarbonyl group as defined above which is
substituted with at least one aryl group having from 6 to 14 carbon atoms
as defined above, examples of which include 4-phenylbenzoyl groups;
[0108] (vi) half-ester salt residues of succinic acid; [0109] (vii)
phosphate ester salt residues; [0110] (viii) ester-forming residues of an
amino acid such as glutamate and aspartate; [0111] (ix) carbamoyl groups
which may optionally be substituted with 1 or 2 alkyl groups having from
1 to 6 carbon atoms as defined above; and [0112] (x)
1-(acyloxy)alkoxycarbonyl groups which comprise a lower alkoxycarbonyl
group as defined above in which the lower alkoxy moiety is substituted
with an aliphatic acyloxy group as defined above or an aromatic acyloxy
group as defined above, examples of which include
pivaloyloxymethyloxycarbonyl groups.

[0113] Of the above protecting groups which are capable of being removed
by a metabolic process (e.g. hydrolysis) in vivo which are used to
synthesise a compound of formula (I), (IV) or (V) in which a hydroxyl
residue therein is modified, the C1-C25 alkylcarbonyl groups
and substituted carbonyloxyalkyl groups are preferred.

[0115] Of the compounds of formula (Ia) and pharmacologically acceptable
salts and esters thereof, preferred are those wherein:

(i) R1 is an alkyl group having from 1 to 4 carbon atoms, an alkenyl
group having from 2 to 4 carbon atoms or an alkynyl group having from 2
to 4 carbon atoms; (ii) R1 is a methyl group or an ethynyl group;
(iii) R2 is hydroxyl, an alkanoyloxy group having from 2 to 5 carbon
atoms, a group of formula (II) wherein n is 0, 1 or 2, and R11 is an
alkyl group having from 1 to 4 carbon atoms, a hydroxyl group, an alkoxy
group having from 1 to 4 carbon atoms or a group of formula
--N(R14)2 wherein each group R14 is the same or different
and is hydrogen or an alkyl group having from 1 to 4 carbon atoms or a
group of formula (III) wherein m is 0, 1 or 2, and each of R12 and
R13 is the same or different and is hydrogen or an alkyl group
having from 1 to 4 carbon atoms; (iv) R2 is hydroxyl, an alkanoyloxy
group having 2 or 3 carbon atoms, a group of formula (II) wherein n is 0
and R11 is a methyl group, an ethyl group, a hydroxyl group, a
methoxy group, an ethoxy group, an amino group, a methylamino group or a
dimethylamino group, or a group of formula (III) wherein m is 0 or 1, and
each of R12 and R13 is the same or different and is hydrogen, a
methyl group or an ethyl group; (v) R2 is hydroxyl; (vi) R3 is
hydrogen or an alkyl group having from 1 to 4 carbon atoms; (vii) R3
is a methyl group; (viii) R4 is hydrogen or an alkyl group having
from 1 to 4 carbon atoms; (ix) R4 is a methyl group; (x) R7,
R8, R9 and R10 together with the carbon atoms to which
they are attached represent a 5- to 7-membered heterocyclic group, said
5- to 7-membered heterocyclic group optionally being substituted with
from 1 to 3 substituents (said substituents are the same or different and
are selected from substituent group α1 defined below), and

[0116] substituent group α1 is a group consisting of a halogen
atom, a hydroxyl group, an amino group, an alkyl group having from 1 to 4
carbon atoms, an alkoxy group having from 1 to 4 carbon atoms, a phenyl
group and a group of formula --N(R16a)2 wherein each group
R16a is the same or different and is hydrogen or an alkyl group
having from 1 to 4 carbon atoms;

(xi) R7, R8, R9 and R10 together with the carbon
atoms to which they are attached represent an isoxazolyl group; and (xii)
each of R5 and R6 is a hydrogen atom or R5 and R6
together represent a single bond.

[0117] In each group of (i) to (ii), (iii) to (v), (vi) to (vii), (viii)
to (ix) and (x) to (xi) compounds of formula (Ia) and pharmacologically
acceptable salts and esters thereof having substituents falling within
the larger numbered group are more preferred.

[0118] The compounds of formula (Ia) and pharmacologically acceptable
salts and esters thereof which are given by an optional combination of
R1 selected from (i) to (ii), R2 selected from (iii) to (v),
R3 selected from (vi) to (vii), R4 selected from (viii) to
(ix), R7, R8, R9 and R10 selected from (x) to (xi)
and R5 and R6 selected from (xia) are also preferred.

[0119] Compounds of formula (Ia) and pharmacologically acceptable salts
and esters thereof having the following combinations are particularly
preferred:

[0120] The most preferred compounds of formula (Ia) and pharmacologically
acceptable salts and esters thereof are danazol and stanozolol and
pharmacologically acceptable salts and esters thereof:

##STR00006##

[0121] Danazol and stanozolol are known synthetic steroid hormones having
antiandrogen activity. Danazol
(17α-pregna-2,4-dien-20-yno[2,3-d]-isoxazol-17β-ol) is a weak
androgen that binds to numerous steroid hormone receptors and blocks the
synthesis of estradiol, progesterone, testosterone and glucocorticoids;
it is known for use as an oral agent employed in the treatment of
endometriosis. Stanozolol
(17-methyl-5α-androstano[3,2-c]pyrazol-17β-ol) is a synthetic
testosterone analogue.

[0122] Computer modelling shows that danazol should cross the blood brain
barrier (data not shown). As far as is known, it has not been reported
prior to the present invention that danazol could do so.

[0124] Of the compounds of formula (IVa) and pharmacologically acceptable
salts and esters thereof, preferred are those wherein:

(xii) each of R18 and R19 is the same or different and is
hydrogen or an alkyl group having from 1 to 4 carbon atoms; (xiii) each
of R18 and R19 is a methyl group;

[0125] (xiv) each of R20, R21 and R24 is the same or
different and is a hydrogen atom or an alkyl group having from 1 to 4
carbon atoms or R21 is a hydrogen atom and R20 and R24
together represent a single bond;

(xv) each of R20, R21 and R24 is a hydrogen atom or
R21 is a hydrogen atom and R20 and R24 together represent
a single bond; (xvi) R17 is hydrogen or an alkyl group having from 1
to 4 carbon atoms; (xvii) R17 is hydrogen; (xviii) R22 is
hydroxyl, an alkanoyloxy group having from 2 to 5 carbon atoms, a group
of formula (II) wherein n is 0, 1 or 2, and R11 is an alkyl group
having from 1 to 4 carbon atoms, a hydroxyl group, an alkoxy group having
from 1 to 4 carbon atoms or a group of formula --N(R14)2
wherein each group R14 is the same or different and is hydrogen or
an alkyl group having from 1 to 4 carbon atoms or a group of formula
(III) wherein m is 0, 1 or 2, and each of R12 and R13 is the
same or different and is hydrogen or an alkyl group having from 1 to 4
carbon atoms; (xix) R22 is hydroxyl, an alkanoyloxy group having 2
or 3 carbon atoms, a group of formula (II) wherein n is 0 and R11 is
a methyl group, an ethyl group, a hydroxyl group, a methoxy group, an
ethoxy group, an amino group, a methylamino group or a dimethylamino
group, or a group of formula (III) wherein m is 0 or 1, and each of
R12 and R13 is the same or different and is hydrogen, a methyl
group or an ethyl group; (xx) R17 and R22 together represent an
oxo group; (xxi) each of R23 and R29 represents a hydrogen atom
or R23 and R29 together represent an epoxy linkage; (xxii)
R25 is hydrogen or an alkyl group having from 1 to 4 carbon atoms,
R26 is a cyano group and R27 and R28 together with the
carbon atom to which they are attached represent a carbonyl group, or

[0126] R25, R26, R27 and R28 together with the carbon
atoms to which they are attached represent a 5- to 7-membered
heterocyclyl group, said 5- to 7-membered heterocyclyl group optionally
being substituted with from 1 to 3 substituents (said substituents are
the same or different and are selected from substituent group
α1 defined above); and

(xxiii) R25 is hydrogen, R26 is a cyano group and R27 and
R28 together with the carbon atom to which they are attached
represent a carbonyl group, or

[0127] R25, R26, R27 and R28 together with the carbon
atoms to which they are attached represent an isoxazolyl group.

[0128] In each group of (xii) to (xiii), (xiv) to (xv), (xvi) to (xvii),
(xviii) to (xix) and (xxii) to (xxiii), compounds of formula (Na) and
pharmacologically acceptable salts and esters thereof having substituents
falling within the larger numbered group are more preferred.

[0129] The compounds of formula (IVa) and pharmacologically acceptable
salts and esters thereof which are given by an optional combination of
R18 and R19 selected from (xii) to (xiii), R20, R21
and R24 selected from (xiv) to (xv), R17 selected from (xvi),
(xvii) and (xx), R22 selected from (xviii) to (xx), R23 and
R29 selected from (xxi) and R25, R26, R27 and
R28 selected from (xxii) to (xxiii) are also preferred.

[0130] Compounds of formula (IVa) and pharmacologically acceptable salts
and esters thereof having the following combinations are particularly
preferred:

(d) R18 and R19=(xii), R20, R21 and R24=(xiv),
R17=(xvi) and R22=(xviii), R23 and R29=(xxi), and
R25, R26, R27 and R28=(xxii); (e) R18 and
R19=(xiii), R20, R21 and R24=(xv), R17=(xvii)
and R22=(xix), R23 and R29=(xxi), and R25, R26,
R27 and R28=(xxiii); (f) R18 and R19=(xii), R20,
R21 and R24=(xiv), R17 and R22 together=(xx),
R23 and R29=(xxi), and R25, R26, R27 and
R28=(xxii); and (g) R18 and R19=(xiii), R20, R21
and R24=(xv), R17 and R22 together=(xx), R23 and
R29=(xxi), and R25, R26, R27 and R28=(xxiii).

[0132] Trilostane
(2α-cyano-4α,5α-epoxyandrostan-17β-ol-3-one) and
derivatives thereof are synthetic steroid hormones having activity in
lowering the blood concentrations of glucocorticoids such as cortisol.
Trilostane is known as an oral medication for the treatment of Cushing's
Syndrome and advanced breast cancer and is described in UK Patent Nos.
1,123,770, 2,130,588 and 2,345,851, U.S. Pat. No. 3,296,295 and
WO-A-02/080930, the contents of which are incorporated herein by
reference thereto.

[0133] Computer modelling shows that trilostane should very readily cross
the blood brain barrier (data not shown). As far as is known, it has not
been reported prior to the present invention that trilostane could do so.

[0135] Of these compounds of formula (Va) and pharmacologically acceptable
salts and esters thereof, preferred are those wherein:

(i) R35 is an alkyl group having from 1 to 4 carbon atoms, an
alkenyl group having from 2 to 4 carbon atoms or an alkynyl group having
from 2 to 4 carbon atoms; (ii) R35 is a methyl group or an ethynyl
group; (iii) R36 is hydroxyl, an alkanoyloxy group having from 2 to
5 carbon atoms, a group of formula (II) wherein n is 0, 1 or 2, and
R11 is an alkyl group having from 1 to 4 carbon atoms, a hydroxyl
group, an alkoxy group having from 1 to 4 carbon atoms or a group of
formula --N(R14)2 wherein each group R14 is the same or
different and is hydrogen or an alkyl group having from 1 to 4 carbon
atoms or a group of formula (III) wherein m is 0, 1 or 2, and each of
R12 and R13 is the same or different and is hydrogen or an
alkyl group having from 1 to 4 carbon atoms; (iv) R36 is hydroxyl,
an alkanoyloxy group having 2 or 3 carbon atoms, a group of formula (II)
wherein n is 0 and R11 is a methyl group, an ethyl group, a hydroxyl
group, a methoxy group, an ethoxy group, an amino group, a methylamino
group or a dimethylamino group, or a group of formula (III) wherein m is
0 or 1, and each of R12 and R13 is the same or different and is
hydrogen, a methyl group or an ethyl group; (v) R36 is hydroxyl;
(vi) R37 is hydrogen or an alkyl group having from 1 to 4 carbon
atoms; (vii) R37 is a methyl group; (viii) R38 is hydrogen or
an alkyl group having from 1 to 4 carbon atoms; (ix) R38 is a methyl
group; (x) each of R41 and R42 is a hydrogen atom and R43
and R44 together with the carbon atom to which they are attached
represent a carbonyl group, or

[0136] R41, R42, R43 and R44 together with the carbon
atoms to which they are attached represent a 5- to 7-membered
heterocyclic group, said 5- to 7-membered heterocyclic group optionally
being substituted with from 1 to 3 substituents (said substituents are
the same or different and are selected from substituent group
α1 defined below), and

[0137] substituent group α1 represents a group consisting of a
halogen atom, a hydroxyl group, an amino group, an alkyl group having
from 1 to 4 carbon atoms, an alkoxy group having from 1 to 4 carbon
atoms, a phenyl group and a group of formula --N(R16a)2 wherein
each group R16a is the same or different and is hydrogen or an alkyl
group having from 1 to 4 carbon atoms;

(xi) R41, R42, R43 and R44 together with the carbon
atoms to which they are attached represent an isoxazolyl group; and (xii)
each of R39 and R40 is a hydrogen atom or R39 and R40
together represent a single bond.

[0138] In each group of (i) to (ii), (iii) to (v), (vi) to (vii), (viii)
to (ix) and (x) to (xi) compounds of formula (Va) and pharmacologically
acceptable salts and esters thereof having substituents falling within
the larger numbered group are more preferred.

[0139] The compounds of formula (Va) and pharmacologically acceptable
salts and esters thereof which are given by an optional combination of
R35 selected from (i) to (ii), R36 selected from (iii) to (v),
R37 selected from (vi) to (vii), R38 selected from (viii) to
(ix), R41, R42, R43 and R44 selected from (x) to (xi)
and R39 and R40 selected from (xia) are also preferred.

[0140] Compounds of formula (Va) and pharmacologically acceptable salts
and esters thereof having the following combinations are particularly
preferred:

[0141] The most preferred compounds of formula (Va) and pharmacologically
acceptable salts and esters thereof are ethisterone, danazol and
stanozolol and pharmacologically acceptable salts and esters thereof:

##STR00010##

[0142] Ethisterone is known a synthetic steroid hormone having
antiandrogen activity. Ethisterone
(17α-hydroxypregn-4-en-20-yn-3-one) is a progestogen that has been
used in the past to treat menstrual disorders and as a component of
combined oral contraceptives. Danazol and stanozolol were discussed
above.

[0143] As noted above, a steroid of formula I or IV, or a
pharmacologically-acceptable salt or ester thereof, can be used to treat
an angiogenic disease or condition of the eye or brain. To do so, a
steroid of formula I or IV, or a pharmacologically-acceptable salt or
ester thereof, is administered to an animal in need of treatment.
Preferably, the animal is a mammal, such as a rabbit, goat, dog, cat,
horse or human. Most preferably, the animal is a human.

[0144] As also noted above, a steroid of formula I, IV or V, or a
pharmacologically-acceptable salt or ester thereof, can be used to treat
an inflammatory disease or condition of the eye or brain. To do so, a
steroid of formula I, IV or V, or a pharmacologically-acceptable salt or
ester thereof, is administered to an animal in need of treatment.
Preferably, the animal is a mammal, such as a rabbit, goat, dog, cat,
horse or human. Most preferably, the animal is a human.

[0145] Effective dosage forms, modes of administration and dosage amounts
for the compounds of the invention may be determined empirically, and
making such determinations is within the skill of the art. It is
understood by those skilled in the art that the dosage amount will vary
with the particular compound employed, the disease or condition to be
treated, the severity of the disease or condition, the route(s) of
administration, the rate of excretion of the compound, the duration of
the treatment, the identify of any other drugs being administered to the
animal, the age, size and species of the animal, and like factors known
in the medical and veterinary arts. In general, a suitable daily dose of
a compound of the present invention will be that amount of the compound
which is the lowest dose effective to produce a therapeutic effect.
However, the daily dosage will be determined by an attending physician or
veterinarian within the scope of sound medical judgment. If desired, the
effective daily dose may be administered as two, three, four, five, six
or more sub-doses, administered separately at appropriate intervals
throughout the day. Administration of the compound should be continued
until an acceptable response is achieved.

[0146] The compounds of the present invention (i.e., steroids of formulas
(I), (IV) and (V) and pharmacologically-acceptable salts and esters
thereof) may be administered to an animal patient for therapy by any
suitable route of administration, including orally, nasally, parenterally
(e.g., intravenously, intraperitoneally, subcutaneously or
intramuscularly), transdermally, intraocularly and topically (including
buccally and sublingually). The preferred routes of administration for
treatment of diseases and conditions of the eye are orally, intraocularly
and topically. Most preferred is topically. The preferred routes of
administration for treatment of diseases and conditions of the brain are
orally and parenterally. Most preferred is orally.

[0147] While it is possible for a compound of the present invention to be
administered alone, it is preferable to administer the compound as a
pharmaceutical formulation (composition). The pharmaceutical compositions
of the invention comprise a compound or compounds of the invention as an
active ingredient in admixture with one or more
pharmaceutically-acceptable carriers and, optionally, with one or more
other compounds, drugs or other materials. Each carrier must be
"acceptable" in the sense of being compatible with the other ingredients
of the formulation and not injurious to the animal.
Pharmaceutically-acceptable carriers are well known in the art.
Regardless of the route of administration selected, the compounds of the
present invention are formulated into pharmaceutically-acceptable dosage
forms by conventional methods known to those of skill in the art. See,
e.g., Remington's Pharmaceutical Sciences.

[0148] Formulations of the invention suitable for oral administration may
be in the form of capsules, cachets, pills, tablets, powders, granules or
as a solution or a suspension in an aqueous or non-aqueous liquid, or an
oil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,
or as pastilles (using an inert base, such as gelatin and glycerin, or
sucrose and acacia), and the like, each containing a predetermined amount
of a compound or compounds of the present invention as an active
ingredient. A compound or compounds of the present invention may also be
administered as bolus, electuary or paste.

[0149] In solid dosage forms of the invention for oral administration
(capsules, tablets, pills, dragees, powders, granules and the like), the
active ingredient (i.e., one or more steroids of the formulas set forth
above and/or pharmacologically-acceptable salts and/or esters thereof) is
mixed with one or more pharmaceutically acceptable carriers, such as
sodium citrate or dicalcium phosphate, and/or any of the following: (1)
fillers or extenders, such as starches, lactose, sucrose, glucose,
mannitol, and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato or
tapioca starch, alginic acid, certain silicates, and sodium carbonate;
(5) solution retarding agents, such as paraffin; (6) absorption
accelerators, such as quaternary ammonium compounds; (7) wetting agents,
such as, for example, cetyl alcohol and glycerol monosterate; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such as
talc, calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In
the case of capsules, tablets and pills, the pharmaceutical compositions
may also comprise buffering agents. Solid compositions of a similar type
may be employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.

[0150] A tablet may be made by compression or molding optionally with one
or more accessory ingredients. Compressed tablets may be prepared using
binder (for example, gelatin or hydroxypropylmethyl cellulose),
lubricant, inert diluent, preservative, disintegrant (for example, sodium
starch glycolate or cross-linked sodium carboxymethyl cellulose),
surface-active or dispersing agent. Molded tablets may be made by molding
in a suitable machine a mixture of the powdered compound moistened with
an inert liquid diluent.

[0151] The tablets, and other solid dosage forms of the pharmaceutical
compositions of the present invention, such as dragees, capsules, pills
and granules, may optionally be scored or prepared with coatings and
shells, such as enteric coatings and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as to
provide slow or controlled release of the active ingredient therein
using, for example, hydroxypropylmethyl cellulose in varying proportions
to provide the desired release profile, other polymer matrices, liposomes
and/or microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter. These compositions may also
optionally contain opacifying agents and may be of a composition that
they release the active ingredient only, or preferentially, in a certain
portion of the gastrointestinal tract, optionally, in a delayed manner.
Examples of embedding compositions which can be used include polymeric
substances and waxes. The active ingredient can also be in
microencapsulated form.

[0155] The invention also provides pharmaceutical products suitable for
treatment of the eye. Such pharmaceutical products include pharmaceutical
compositions, devices and implants (which may be compositions or
devices).

[0156] Pharmaceutical formulations (compositions) for intraocular
injection of a compound or compounds of the invention into the eyeball
include solutions, emulsions, suspensions, particles, capsules,
microspheres, liposomes, matrices, etc. See, e.g., U.S. Pat. No.
6,060,463, U.S. Patent Application Publication No. 2005/0101582, and PCT
application WO 2004/043480, the complete disclosures of which are
incorporated herein by reference. For instance, a pharmaceutical
formulation for intraocular injection may comprise one or more compounds
of the invention in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous
solutions, suspensions or emulsions, which may contain antioxidants,
buffers, suspending agents, thickening agents or viscosity-enhancing
agents (such as a hyaluronic acid polymer). Examples of suitable aqueous
and nonaqueous carriers include water, saline (preferably 0.9%), dextrose
in water (preferably 5%), buffers, dimethylsulfoxide, alcohols and
polyols (such as glycerol, propylene glycol, polyethylene glycol, and the
like). These compositions may also contain adjuvants such as wetting
agents and emulsifying agents and dispersing agents. In addition,
prolonged absorption of the injectable pharmaceutical form may be brought
about by the inclusion of agents which delay absorption such as polymers
and gelatin. Injectable depot forms can be made by incorporating the drug
into microcapsules or microspheres made of biodegradable polymers such as
polylactide-polyglycolide. Examples of other biodegradable polymers
include poly(orthoesters), poly(glycolic) acid, poly(lactic) acid,
polycaprolactone and poly(anhydrides). Depot injectable formulations are
also prepared by entrapping the drug in liposomes (composed of the usual
ingredients, such as dipalmitoyl phosphatidylcholine) or microemulsions
which are compatible with eye tissue. Depending on the ratio of drug to
polymer or lipid, the nature of the particular polymer or lipid
components, the type of liposome employed, and whether the microcapsules
or microspheres are coated or uncoated, the rate of drug release from
microcapsules, microspheres and liposomes can be controlled.

[0157] The compounds of the invention can also be administered surgically
as an ocular implant. For instance, a reservoir container having a
diffusible wall of polyvinyl alcohol or polyvinyl acetate and containing
a compound or compounds of the invention can be implanted in or on the
sclera. As another example, a compound or compounds of the invention can
be incorporated into a polymeric matrix made of a polymer, such as
polycaprolactone, poly(glycolic) acid, poly(lactic) acid,
poly(anhydride), or a lipid, such as sebacic acid, and may be implanted
on the sclera or in the eye. This is usually accomplished with the animal
receiving a topical or local anesthetic and using a small incision made
behind the cornea. The matrix is then inserted through the incision and
sutured to the sclera.

[0158] A preferred embodiment of the invention is local topical
administration of the compounds of the invention to the eye, and a
particularly preferred embodiment of the invention is a topical
pharmaceutical composition suitable for application to the eye. Topical
pharmaceutical compositions suitable for application to the eye include
solutions, suspensions, dispersions, drops, gels, hydrogels and
ointments. See, e.g., U.S. Pat. No. 5,407,926 and PCT applications WO
2004/058289, WO 01/30337 and WO 01/68053, the complete disclosures of all
of which are incorporated herein by reference.

[0159] Topical formulations suitable for application to the eye comprise
one or more compounds of the invention in an aqueous or nonaqueous base.
The topical formulations can also include absorption enhancers,
permeation enhancers, thickening agents, viscosity enhancers, agents for
adjusting and/or maintaining the pH, agents to adjust the osmotic
pressure, preservatives, surfactants, buffers, salts (preferably sodium
chloride), suspending agents, dispersing agents, solubilizing agents,
stabilizers and/or tonicity agents. Topical formulations suitable for
application to the eye will preferably comprise an absorption or
permeation enhancer to promote absorption or permeation of the compound
or compounds of the invention into the eye and/or a thickening agent or
viscosity enhancer that is capable of increasing the residence time of a
compound or compounds of the invention in the eye. See PCT applications
WO 2004/058289, WO 01/30337 and WO 01/68053. Exemplary
absorption/permeation enhancers include methysulfonylmethane, alone or in
combination with dimethylsulfoxide, carboxylic acids and surfactants.
Exemplary thickening agents and viscosity enhancers include dextrans,
polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels,
Gelrite®, cellulosic polymers (such as hydroxypropyl
methylcellulose), carboxyl-containing polymers (such as polymers or
copolymers of acrylic acid), polyvinyl alcohol and hyaluronic acid or a
salt thereof.

[0160] Liquid dosage forms (e.g., solutions, suspensions, dispersions and
drops) suitable for treatment of the eye can be prepared, for example, by
dissolving, dispersing, suspending, etc. a compound or compounds of the
invention in a vehicle, such as, for example, water, saline, aqueous
dextrose, glycerol, ethanol and the like, to form a solution, dispersion
or suspension. If desired, the pharmaceutical formulation may also
contain minor amounts of non-toxic auxiliary substances, such as wetting
or emulsifying agents, pH buffering agents and the like, for example
sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate,
triethanolamine oleate, etc.

[0161] Aqueous solutions and suspensions suitable for treatment of the eye
can include, in addition to a compound or compounds of the invention,
preservatives, surfactants, buffers, salts (preferably sodium chloride),
tonicity agents and water. If suspensions are used, the particle sizes
should be less than 10 μM to minimize eye irritation. If solutions or
suspensions are used, the amount delivered to the eye should not exceed
50 μl to avoid excessive spillage from the eye.

[0162] Colloidal suspensions suitable for treatment of the eye are
generally formed from microparticles (i.e., microspheres, nanospheres,
microcapsules or nanocapsules, where microspheres and nanospheres are
generally monolithic particles of a polymer matrix in which the
formulation is trapped, adsorbed, or otherwise contained, while with
microcapsules and nanocapsules the formulation is actually encapsulated).
The upper limit for the size of these microparticles is about 5μ to
about 10μ.

[0163] Ophthalmic ointments suitable for treatment of the eye include a
compound or compounds of the invention in an appropriate base, such as
mineral oil, liquid lanolin, white petrolatum, a combination of two or
all three of the foregoing, or polyethylene-mineral oil gel. A
preservative may optionally be included.

[0164] Ophthalmic gels suitable for treatment of the eye include a
compound or compounds of the invention suspended in a hydrophilic base,
such as Carpobol-940 or a combination of ethanol, water and propylene
glycol (e.g., in a ratio of 40:40:20). A gelling agent, such as
hydroxylethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose or ammoniated glycyrrhizinate, is used. A
preservative and/or a tonicity agent may optionally be included.

[0165] Hydrogels suitable for treatment of the eye are formed by
incorporation of a swellable, gel-forming polymer, such as those listed
above as thickening agents or viscosity enhancers, except that a
formulation referred to in the art as a "hydrogel" typically has a higher
viscosity than a formulation referred to as a "thickened" solution or
suspension. In contrast to such preformed hydrogels, a formulation may
also be prepared so to form a hydrogel in situ following application to
the eye. Such gels are liquid at room temperature but gel at higher
temperatures (and thus are termed "thermoreversible" hydrogels), such as
when placed in contact with body fluids. Biocompatible polymers that
impart this property include acrylic acid polymers and copolymers,
N-isopropylacrylamide derivatives and ABA block copolymers of ethylene
oxide and propylene oxide (conventionally referred to as "poloxamers" and
available under the Pluronic® tradename from BASF-Wayndotte).

[0166] Preferred dispersions are liposomal, in which case the formulation
is enclosed within liposomes (microscopic vesicles composed of
alternating aqueous compartments and lipid bilayers).

[0167] Eye drops can be formulated with an aqueous or nonaqueous base also
comprising one or more dispersing agents, solubilizing agents or
suspending agents. Drops can be delivered by means of a simple eye
dropper-capped bottle or by means of a plastic bottle adapted to deliver
liquid contents dropwise by means of a specially shaped closure.

[0168] The compounds of the invention can also be applied topically by
means of drug-impregnated solid carrier that is inserted into the eye.
Drug release is generally effected by dissolution or bioerosion of the
polymer, osmosis, or combinations thereof. Several matrix-type delivery
systems can be used. Such systems include hydrophilic soft contact lenses
impregnated or soaked with the desired compound of the invention, as well
as biodegradable or soluble devices that need not be removed after
placement in the eye. These soluble ocular inserts can be composed of any
degradable substance that can be tolerated by the eye and that is
compatible with the compound of the invention that is to be administered.
Such substances include, but are not limited to, poly(vinyl alcohol),
polymers and copolymers of polyacrylamide, ethylacrylate and
vinylpyrrolidone, as well as cross-linked polypeptides or
polysaccharides, such as chitin.

[0169] Dosage forms for the other types of topical administration (i.e.,
not to the eye) or for transdermal administration of compounds of the
invention include powders, sprays, ointments, pastes, creams, lotions,
gels, solutions, patches, drops and inhalants. The active ingredient may
be mixed under sterile conditions with a pharmaceutically-acceptable
carrier, and with any buffers, or propellants which may be required. The
ointments, pastes, creams and gels may contain, in addition to the active
ingredient, excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or
mixtures thereof. Powders and sprays can contain, in addition to the
active ingredient, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder or mixtures of
these substances. Sprays can additionally contain customary propellants
such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane. Transdermal patches have the added advantage
of providing controlled delivery of compounds of the invention to the
body. Such dosage forms can be made by dissolving, dispersing or
otherwise incorporating one or more compounds of the invention in a
proper medium, such as an elastomeric matrix material. Absorption
enhancers can also be used to increase the flux of the compound across
the skin. The rate of such flux can be controlled by either providing a
rate-controlling membrane or dispersing the compound in a polymer matrix
or gel.

[0170] Pharmaceutical formulations include those suitable for
administration by inhalation or insufflation or for nasal administration.
For administration to the upper (nasal) or lower respiratory tract by
inhalation, the compounds of the invention are conveniently delivered
from an insufflator, nebulizer or a pressurized pack or other convenient
means of delivering an aerosol spray. Pressurized packs may comprise a
suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or
other suitable gas. In the case of a pressurized aerosol, the dosage unit
may be determined by providing a valve to deliver a metered amount.

[0171] Alternatively, for administration by inhalation or insufflation,
the composition may take the form of a dry powder, for example, a powder
mix of one or more compounds of the invention and a suitable powder base,
such as lactose or starch. The powder composition may be presented in
unit dosage form in, for example, capsules or cartridges, or, e.g.,
gelatin or blister packs from which the powder may be administered with
the aid of an inhalator, insufflator or a metered-dose inhaler.

[0172] For intranasal administration, compounds of the invention may be
administered by means of nose drops or a liquid spray, such as by means
of a plastic bottle atomizer or metered-dose inhaler. Liquid sprays are
conveniently delivered from pressurized packs. Typical of atomizers are
the Mistometer (Wintrop) and Medihaler (Riker).

[0173] Nose drops may be formulated with an aqueous or nonaqueous base
also comprising one or more dispersing agents, solubilizing agents or
suspending agents. Drops can be delivered by means of a simple eye
dropper-capped bottle or by means of a plastic bottle adapted to deliver
liquid contents dropwise by means of a specially shaped closure.

[0174] Pharmaceutical compositions of this invention suitable for
parenteral administrations comprise one or more compounds of the
invention in combination with one or more pharmaceutically-acceptable
sterile isotonic aqueous or non-aqueous solutions, dispersions,
suspensions or emulsions, or sterile powders which may be reconstituted
into sterile injectable solutions or dispersions just prior to use, which
may contain antioxidants, buffers, solutes which render the formulation
isotonic with the blood of the intended recipient or suspending or
thickening agents.

[0175] Examples of suitable aqueous and nonaqueous carriers which may be
employed in the pharmaceutical compositions of the invention include
water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl oleate.
Proper fluidity can be maintained, for example, by the use of coating
materials, such as lecithin, by the maintenance of the required particle
size in the case of dispersions, and by the use of surfactants.

[0176] These compositions may also contain adjuvants such as wetting
agents, emulsifying agents and dispersing agents. It may also be
desirable to include isotonic agents, such as sugars, sodium chloride,
and the like in the compositions. In addition, prolonged absorption of
the injectable pharmaceutical form may be brought about by the inclusion
of agents which delay absorption such as aluminum monosterate and
gelatin.

[0177] In some cases, in order to prolong the effect of a drug, it is
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This may be accomplished by the use of a liquid
suspension of crystalline or amorphous material having poor water
solubility. The rate of absorption of the drug then depends upon its rate
of dissolution which, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally-administered drug is accomplished by dissolving or
suspending the drug in an oil vehicle.

[0178] Injectable depot forms are made by forming microencapsule matrices
of the drug in biodegradable polymers such as polylactide-polyglycolide.
Depending on the ratio of drug to polymer, and the nature of the
particular polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared by
entrapping the drug in liposomes or microemulsions which are compatible
with body tissue. The injectable materials can be sterilized for example,
by filtration through a bacterial-retaining filter.

[0179] The formulations may be presented in unit-dose or multi-dose sealed
containers, for example, ampules and vials, and may be stored in a
lyophilized condition requiring only the addition of the sterile liquid
carrier, for example water for injection, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from
sterile powders, granules and tablets of the type described above.

[0180] Additional objects, advantages and novel features of the present
invention will become apparent to those skilled in the art by
consideration of the following non-limiting examples.

[0190] 1. Trypsinized HUVEC cells from flasks grown in Cambrex EGM-2 media
to 70-80% confluence were washed two times with 37° C. EGM-2 with
0.1% FCS. 2. Cell suspensions containing 30,000 cells and compounds in
EGM-2 0.1% FCS were added to the upper chamber of inserts. 3. EGM-2
containing 5% FCS was added to the bottom chamber and then incubated for
24 hours at 37° C. and 5% CO2. 4. Non-invading cells were
removed from the upper chamber with cotton swab and the inserts were
washed two times with 37° C. HBSS. 5. Inserts were then placed in
wells containing 10 μM Calcein AM in HBSS. 6. Following four hours at
37° C. and 5% CO2, fluorescence was measured at 485 nm
excitation and 595 nm emission.

Results and Observations:

[0191] Results are presented as mean fluorescent units (FU) in triplicate
(n=3) with background fluorescence subtracted. HUVEC 7016 cells were used
for this experiment exhibiting 95% viability by trypan blue exclusion at
time of seeding. To determine background invasion, nil inserts were
included in triplicate that had EGM-2 with 0.1% FCS added to the bottom
chamber. Without a chemotactic signal, these inserts will give a
background invasion to compare the FCS and FCS plus compound wells. The
results are as shown in Table 1 below and depicted graphically in FIG. 1.

[0192] The high dose of trilostane III dropped the levels back to
background. LY294002, a known inhibitor of invasion, was included as a
control and exhibited similar inhibition as trilostane. LY303511 is an
inactive variant of the LY294002 and had no effect on invasion as
expected. In conclusion, it would seem that trilostane III has an effect
on endothelial cell invasion in certain individuals or at the appropriate
time in the cell cycle.

Example 2

Trilostane III Effect on HUVEC Cell Proliferation

Purpose:

[0193] To determine the effect trilostane III has on HUVEC cell
proliferation.

[0201] 1. HUVEC cells were plated in 96 well plates at 5,000
cells/cm2 and incubated for 24 hours at 37° C. and 5%
CO2 in EGM-2 media. 2. Medium was aspirated and the cells were then
washed two times with 37° C. HBSS. 3. EGM-2 containing 5% FCS with
and without the compound (0.01 μM-200 μM trilostane III) was added
to the wells and incubated for 24, 48, or 72 hours. 4. Cells were again
washed to times with warm HBSS and then Celltiter reagent in EGM-2 with
0.1% FCS was added. 5. After 4 hours in culture the OD of each well was
determine at 470 nm. 6. Repeat steps 4 and 5 for each time point.

Results and Observations:

[0202] Results are presented as mean OD of samples performed in triplicate
(n=3) with mean blank OD subtracted. HUVEC 8750 cells were used for this
experiment with 98% viability by trypan blue exclusion at time of
seeding. Extra fetal calf serum (2% increased to 5%) was added to keep
trilostane III in solution. The raw data are shown in Table 2 below and
depicted graphically in FIG. 2.

[0203] Trilostane III proved effective at inhibiting endothelial cell
proliferation. Concentrations as low as 1 μM (at 24 hours), exhibited
statistically relevant decreases in cell proliferation. Increasing the
dose to 100 μM, inhibited cultures by 33, 67, and 90% at 24, 48, and
72 hours respectively. The initial seeding of 1,500 cells per well is not
detectable by the celltiter assay. Cells must expand to detectable levels
so 100% inhibition can be expected and would not infer cell toxicity.
Viable cells were visible in all wells examined under the inverted
microscope, even at the highest doses after 72 hours in culture. These
results indicate that trilostane III may be an effective antiangiogenic
compound, by interfering with the initial proliferation of endothelial
cells.

Example 3

Trilostane III Effect on HUVEC Angiogenesis: Tube Formation

Purpose:

[0204] To examine the effect of trilostane III on the formation of
tube-like structures by HUVEC cells in an extracellular matrix gel.

[0211] 1. Trypsinized HUVEC cells from flasks grown in Cambrex EGM-2 media
to 70-80% confluence were washed two times with 37° C. EGM-2 with
0.1% FCS. 2. Cell suspensions containing 10,000 cells and compounds in
both EGM-2 0.1% and 5% FCS were added per well then incubated for 18
hours at 37° C. and 5% CO2. 3. Following incubation, tube
formations were photographed under microscope.

Results and Observations:

[0212] Passage 3 HUVEC 8750 cells were used for this experiment at 98%
viability by trypan blue exclusion at time of seeding. Few cells were
obtained for this experiment but did not seem to interfere with the
development of tubes. Pictures of representative wells for the compound
and controls are shown in FIG. 3.

Discussion and Conclusion:

[0213] The final step of angiogenesis is the formation of new vascular
structures. HUVEC cells when grown in gels consisting of extracelluler
matrix proteins, will exhibit a "latticework" of vacuoles that mimic the
inner lumen of the capillary. Addition of fetal calf serum, or other
angiogenic substances, will enhance the length and definition of these
structures. Dosing with 50 μM trilostane III led to a decrease in
branching, vacuole formation, and increase in satellite cells. 50 μM
LY294002, a PI3 kinase inhibitor known to interfere with tube formation,
completely inhibited the development seen in the nil wells while the
inactive form exhibited comparable tube formations to untreated cells.
The inclusion of 5% fetal calf serum to the wells increased tube
definition and vacuole formation. 50 μM trilostane III and 50 μm
LY294002 treatment greatly reduced tube formation in the presence of
fetal calf serum. The untreated cells are far more susceptible to the
effects of the control compounds and trilostane III. In conclusion,
trilostane III appears to prohibit tube formation of HUVEC cells.

Example 4

Danazol Effect on HUVEC Cell Proliferation

Protocol:

[0214] Primary HUVEC and EGM-2 growth medium were obtained from Cambrex
(Walkersville, Md.). The cells were passaged in medium supplemented with
2% fetal calf serum (FCS) in tissue culture flasks at 37° C. and
5% CO2. Subculturing was performed using trypsin when 60-80%
confluence was obtained as specified by the supplier.

[0215] Cryopreserved ampoules of passage 2 HUVEC cells were thawed and
plated in 96 well tissue culture plates at 5,000 cells/cm2. A 50 mM
stock solution of danazol was prepared in ethanol and the FCS in the
medium was increased to 5% to keep danazol in solution. The cells were
treated with medium containing final concentrations of danazol ranging
from 0.1 to 100 μM in triplicates. 24, 48, and 72 hour incubations
were performed and cell proliferation was determined utilizing Celltiter
96 AQueous One Solution Cell Proliferation assay from Promega
(Madison, Wis.). In short, medium was aspirated from each well and the
cells were washed with 200 μl Hepes buffered saline (HBSS) from
Cambrex warmed to 37° C. 100 μl diluted celltiter solution (15
μl stock+85 μl EGM-2 containing 0.1% FCS) were added to each well
and incubated for an additional 4 hours. Optical density was determined
by microplate reader using a 530 nm filter after blank subtraction and
data presented as OD±standard deviation. The final concentration of
ethanol in the wells was less then 0.2% and had no effect on cell
proliferation or viability.

[0216] All data are presented as representative experiment done in
triplicate. Differences between subsets were analyzed using student
t-test in Microsoft Excel. P<0.05 was considered statistically
significant.

Results, Observations and Discussion:

[0217] Culturing primary HUVEC endothelial cells in the presence of
danazol decreased the OD obtained from the Promega celltiter
proliferation assay in a time and dose dependent fashion (FIG. 4). The
celltiter assay is based on the reduction of the assay solution by
dehydrogenase enzymes to a formazan dye that directly correlates to cell
number. Danazol treatment at 24 hours seemed to be effective only at very
high doses. Significant decreases (p value <0.05) in assay OD were
seen at 10 μM or greater concentrations of danazol. The OD detected in
the nil wells was 0.414±0.06 and treatment with 10 μM danazol
decreased the OD to 0.288±0.037 while 100 μM to 0.162±0.017,
equating to percent inhibitions of 30% and 65% respectively. At 48 hours,
the inhibition observed was significant even at physiological levels or
approximately 1 μM. The nil reading obtained after 48 hours in culture
increased to 0.629±0.095 and was reduced to 0.378±0.037 by 1 μM,
0.241±0.012 by 10 μM, and 0.19±0.033 by 100 μM (or percent
inhibitions of 40%, 61%, and 70% respectively). After 72 hours, all
danazol treatments tested exhibited significant reduction in HUVEC
proliferation. The OD obtained in nil wells was 1.113±0.054 and after
0.1 μM treatment fell to 0.798±0.037, 1 μM to 0.484±0.022, 10
μM to 0.229±0.016, and 100 μM to 0.156±0.018 (inhibitions of
28%, 57%, 80%, and 86% respectively). Examination of the OD obtained from
all 100 μM danazol doses was consistent at all time points indicating
a complete arrest of cell proliferation at this concentration. In
summary, danazol exhibited strong inhibition of endothelial cell
proliferation.

Example 5

Danazol Effect on HUVEC Angiogenesis: Tube Formation

Protocol:

[0218] To investigate the formation of capillary-like structures by HUVEC
cells, the Angiogenesis System Endothelial Cell Tube Formation Assay was
purchased from BD Biosciences (San Jose, Calif.) and used according to
the manufacturers protocol. In brief, 100,000 HUVEC cells were seeded
onto rehydrated matrigel plugs in 96 well tissue culture plates in the
presence of 5% FCS to induce tube formation. Danazol was added to final
concentrations of 1 μM, 10 μM, or 100 μM and LY294002 was added
at 100 μM. After 18 hours the wells were photographed using a Kodak
DCS Pro SLR/N digital camera (Rochester, N.Y.) mounted on an inverted
microscope. Ethanol treated wells were included to determine if the
vehicle had any effects on cell differentiation.

Results, Observations and Discussion:

[0219] To elucidate if danazol can prevent the formation of tube-like
structures by HUVEC, 96 well plates containing matrigel plugs were used.
Endothelial cells when cultured in the presence of angiogenic substances
and supplied with an extracellular matrix scaffold will differentiate
into structures loosely resembling capillary vessels. HUVEC cells grown
with danazol exhibited fewer organized structures with thin and less
defined interconnections than controls (see FIG. 5, in which A=control,
B=1 μM danazol, C=10 μM danazol, D=50 μM danazol, and E=50 μM
LY294002). Treatment with 50 μM danazol led to isolated colonies of
HUVEC located in the plug with very few, thin connections or vessel lumen
spaces. The effect of danazol was very similar to the positive control
compound LY294002. To ensure that the vehicle used had no effect, wells
were treated with ethanol at concentrations corresponding to the highest
dose of danazol used and no effect on tube formation was observed (data
not shown). Thes data indicate that danazol is an effective inhibitor of
tube formation.

Example 6

Danazol Effect on HUVEC Angiogenesis: Invasion Chamber

Protocol:

[0220] BioCoat Matrigel Invasion Chambers were purchased from BD
Biosciences (San Jose, Calif.). Inserts were rehydrated at 37° C.
with 500 μl HBSS for 2 hours prior to use in humidified incubator.
Trypsinized HUVEC cells were washed twice with warm EGM-2 containing 0.1%
FCS and added to the upper chamber of the invasion insert at 100,000
cells in a total volume of 250 μl. Danazol and control compounds were
added to the upper reservoir at final concentrations of 10 μM and 100
μM. 750 μl EGM-2 supplemented with 5% FCS was added to the bottom
chamber to initiate invasion and the plates were incubated for 24 hours.
Non-invasive cells were removed from the upper chamber with moistened
cotton swabs and then the inserts were washed twice with HBSS. The
inserts were then submerged in 10 μM calcein AM prepared in HBSS and
incubated for 4 hours. Fluorescence was determined in a microplate reader
at 485 nm excitation and 595 nm emission. LY294002 and the structurally
similar but inactive compound LY303511 served as positive and negative
controls respectively for this experiment.

Results:

[0221] The results are presented in FIG. 6. All data is presented as
representative experiment done in triplicate. Differences between subsets
were analyzed using student t-test in Microsoft Excel. P<0.05 was
considered statistically significant.

[0222] Porous, matrigel coated inserts were used to determine if danazol
can interfere with the invasion or migration of endothelial cells (FIG.
6). In the system used for our study, a significant increase in cells was
detected by fluorescent dye after the addition of FCS to the chamber
opposite the endothelial cells (5674 FU±77 to 7143±516). Danazol at
concentrations of 10 μM and 100 μM had no effect, while LY294002
showed almost complete attenuation of cell invasion (5814±153). These
data indicate that factors present in the FCS induce the production of
proteases that digest extracellular matrix by HUVEC cells followed by
migration along a chemotactic gradient. Danazol has no apparent
inhibitory effect on invasion and migration of HUVEC cells in this model.